1. Field of the Invention
The present invention relates to microfluidic cell culture systems and, more specifically, to microfluidic cell culture systems comprising microporous, polymeric, flat or three-dimensional membranes that can mimic barrier tissue. These membranes can be incorporated into, and fabricated simultaneously with, a microfluidic chamber. Accordingly, the fabrication allows for leak-free, direct attachment of the microporous membranes to the walls of the microfluidic chamber.
2. Description of the Related Art
Microfluidic cell culture systems that contain several compartments for the culture of multiple tissues such as the gastrointestinal tract epithelium, liver, kidney and tumor tissues are currently being developed for the purpose of studying the collective response of multiple organs to new drugs under near physiologic conditions. Low bioavailability of drugs at the target organ has been cited as one of the major reasons for the failure of newly developed drugs in clinical trials. Drug testing systems that contain models of barrier tissues such as the gastrointestinal tract epithelium and the lung epithelium are invaluable for correctly predicting the bioavailability of newly developed drugs early in the drug development process.
Microfluidic tissue analogs of barrier tissues replicate the physiologic aspects of these tissues with more authenticity than static models that are currently being used. In microfluidic systems the ratios of in vivo masses or volumes of organs can be recreated on chip, and physiologic fluid residence times in each organ can be achieved. Moreover, metabolites produced in one tissue can travel to other organ compartments and affect the tissues cultured there. Such devices have the potential to improve the drug screening process significantly, because they recreate parts of the human metabolism.
A few microfluidic in vitro analogs of barrier tissues have been developed so far, but none of these models recreate the three dimensional villi structure that increases the absorptive area in the small intestine. The existing models also do not combine other tissues with the developed models in one system. The developed devices may contain semipermeable membranes that were sandwiched between two microfluidic chambers. The membranes provide a surface for cell growth and at the same time they separate the chambers that contain differing concentrations of compounds that are used in the study. The pores in the membranes allow for the transport of molecules across the cell layer. Using semipermeable membranes, the development of in vitro cell culture analogs of tissues of the lung, the gastrointestinal tract and the kidney has been achieved. However, sandwiching membranes between two existing tissue chambers is challenging. It is not always possible to assemble such systems so that they are leak-free.
Because sheets of membranes are difficult to integrate with microfluidic systems, multi-organ cell culture devices are limited in regards to the barrier tissues they contain. As a consequence the microfluidic systems that connect the basolateral chamber of a barrier tissue model with another tissue chamber utilize off-chip modules that are connected to on-chip components. For example, others have developed off-chip chambers that utilize transwell membrane inserts for the culture of intestinal epithelial cells under fluidic conditions. Both systems have been used in combination with liver cell chambers to create in vitro models of intestinal absorption and first pass metabolism of drugs. Both systems cannot scale the surface area of the intestinal epithelium to approximate the in vivo ratio of epithelial surface area to the mass and volume of liver tissue because the sizes of transwell membranes that are used within the modules are fixed. Membranes that are fabricated directly on top of microfluidic chambers and that can be directly attached to the chamber sidewalls could be scaled appropriately and would not be prone to leaking.
Porous silicon nitride membranes address one of these issues because they can span microfluidic compartments of varying sizes and have been used for the culture of endothelial cells in an effort to recreate part of the blood brain barrier in vitro. However, silicon nitride membranes are typically patterned at the front side of silicon wafers and released by etching from the backside. Integrating these membranes with microfluidic systems requires one or two bonding steps to create closed fluidic chambers. This process is not straightforward if the desired chamber depth is less than the thickness of the silicon substrate (˜500 μm), which is typically the case in “body-on-a-chip” devices. Flexibility in choosing chamber dimensions such as the chamber depth is necessary to be able to adjust fluid residence times within individual organ compartments. Typical values for chamber depths range from 20-200 μm.
In addition, both commercially available membranes and microfabricated membranes are typically flat and do not recreate the three-dimensional aspects of tissues such as the macro villi of the gastrointestinal tract epithelium. The gastrointestinal tract epithelium contains both micro and macro villi that increase the absorptive area of the intestine. Conventional models of the gastrointestinal tract such as Caco-2 monolayers do not provide three-dimensional surfaces that take the geometric character of the tissue into account. Mimicking the tissue organization of the intestinal epithelium authentically requires microscale three-dimensional semipermeable membranes that act as cell culture surface and control mass transport.
Other prefabricated membranes also do not mimic the appropriate three-dimensional structure of tissue, (that means they are not three-dimensional) because they either lack the required rigidity to control a 3-D three-dimensional and/or lack sufficient porosity. Any three-dimensional cell culture scaffold to date is not a membrane. They are based on alginate, basement membrane proteins, plastics or polymerizable compounds. Accordingly, access to the underside of the cell culture is not possible with these methods.